Research Article |
Corresponding author: Zhi-Yuan Zhang ( zhangzhiyuan_16@163.com ) Corresponding author: Yan-Feng Han ( swallow1128@126.com ) Academic editor: Thorsten Lumbsch
© 2023 Zhi-Yuan Zhang, Yao Feng, Shuo-Qiu Tong, Chen-Yu Ding, Gang Tao, Yan-Feng Han.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Zhang Z-Y, Feng Y, Tong S-Q, Ding C-Y, Tao G, Han Y-F (2023) Morphological and phylogenetic characterisation of two new soil-borne fungal taxa belonging to Clavicipitaceae (Hypocreales, Ascomycota). MycoKeys 98: 113-132. https://doi.org/10.3897/mycokeys.98.106240
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The fungal taxa belonging to the Clavicipitaceae (Hypocreales, Ascomycota) are widely distributed and include diverse saprophytic, symbiotic and pathogenic species that are associated with soils, insects, plants, fungi and invertebrates. In this study, we identified two new fungal taxa belonging to the family Clavicipitaceae that were isolated from soils collected in China. Morphological characterisation and phylogenetic analyses showed that the two species belong to Pochonia (Pochonia sinensis sp. nov.) and a new genus for which we propose Paraneoaraneomyces gen. nov. in Clavicipitaceae.
Clavicipitaceae, entomopathogenic fungi, new taxa, phylogeny, Pochonia, taxonomy
Fungi are found in a wide array of ecological niches and play key roles as decomposers, mutualists and pathogens (
Phylogenetic analyses showed that the Verticillium section Prostrata was heterogenous and Pochonia was recognised as a distinct genus with several species that often form dictyochlamydospores and were parasitic on the nematode cysts and eggs (
In this study, we report the morphological and phylogenetic characterisation of two new taxa belonging to the family Claviciptaceae that were isolated from the urban soil samples in China.
The soil samples were collected in June 2020 from the Cengong County (27°16’98’’N, 108°81’46’’E) in Kaili City, Guizhou Province, China. The fungi were isolated using the methods described previously (
The phenotype was determined by growing the single isolates in plates containing potato dextrose agar (PDA), malt extract agar (MEA), oatmeal agar (OA) and synthetic low-nutrient agar (SNA) medium. The plates were incubated in the dark at 25 °C for 14 days. The photomicrographs of the fungal structures were obtained using an OLYMPUS BX53 microscope equipped with differential interference contrast (DIC) optics, an OLYMPUS DP73 high-definition colour camera and the cellSens software version 1.18.
Total DNA was extracted using the 5% chelex-100 solution as described previously (
Molecular marker | Primer name | Primer sequence (5´-3´) | Reference |
SSU | NS1 | GTAGTCATATGCTTGTCTC |
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NS4 | CTTCCGTCAATTCCTTTAAG |
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ITS | ITS1 | TCCGTAGGTGAACCTGCG |
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ITS4 | TCCTCCGCTTATTGATATGC |
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LSU | LR0R | ACCCGCTGAACTTAAGC |
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LR7 | TACTACCACCAAGATCT |
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EF1A | 2218R | ATGACACCRACRGCRACRGTYTG |
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983F | GCYCCYGGHCAYCGTGAYTTYAT |
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RPB2 | fRPB2-5F | GAYGAYMGWGATCAYTTYGG |
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RPB2-7cR | CCCATRGCTTGYTTRCCCAT |
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Species | Strains | SSU | ITS | LSU | RPB2 | EF1A | References |
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Aciculosporium oplismeni | MAFF 246966 | – | LC571760 | LC571760 | LC572054 | LC572040 |
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Aciculosporium take | MAFF 241224 | – | LC571753 | LC571753 | LC572048 | LC572034 |
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TNS-F-60465 | – | LC571755 | LC571756 | LC572049 | LC572035 |
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Aschersonia confluens | BCC 7961 | – | JN049841 | DQ384947 | DQ452465 | DQ384976 |
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Aschersonia placenta | BCC 7869 | EF469121 | JN049842 | EF469074 | EF469104 | EF469056 |
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Atkinsonella hypoxylon | B4728 | – | – | – | KP689514 | KP689546 |
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Balansia epichloe | A.E.G. 96-15a | – | JN049848 | – | EF468908 | EF468743 |
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Balansia henningsiana | A.E.G. 96-27a | AY545723 | JN049815 | AY545727 | DQ522413 | AY489610 |
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Claviceps fusiformis | ATCC 26019 | DQ522539 | JN049817 | U17402 | – | DQ522320 | Rehner et al. (1995); |
Claviceps purpurea | GAM 12885 | – | U57669 | AF543789 | DQ522417 | AF543778 |
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SA cp 11 | EF469122 | EF469075 | EF469105 | EF469058 |
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Collarina aurantiaca | FMR 11134 | – | KJ807178 | KJ807181 | – | – |
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FMR 11784 | – | KJ807177 | KJ807180 | – | – |
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Conoideocrella luteorostrata | NHJ 11343 | EF468995 | JN049859 | – | – | EF468801 |
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NHJ 12516 | EF468994 | JN049860 | – | EF468946 | EF468800 |
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Conoideocrella tenuis | NHJ 6293 | EU369112 | JN049862 | EU369044 | EU369087 | EU369029 |
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Corallocytostroma ornithocopreoides | WAC 8705 | – | – | – | LT216620 | LT216546 |
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Dussiella tuberiformis | J.F.White | – | – | – | JQ257020 | JQ257027 |
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Ephelis japonica | CBS 236.64 | – | MH858427 | – | – | – |
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Eph.oryzae | – | AB038564 | – | – | – |
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Ephelis tripsaci | CBS 857.72 | – | KP859042 | KP858978 | – | – |
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Epichloë elymi | C.Schardl 760 | – | – | AY986924 | – | AY986951 |
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Epichloë typhina | ATCC 56429 | – | JN049832 | U17396 | DQ522440 | AF543777 | Rehner et al. (1995); |
Helicocollum surathaniense | BCC 34463 | – | – | KT222328 | – | KT222336 |
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BCC 34464 | – | – | KT222329 | – | KT222337 |
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Heteroepichloe bambusae | Ba-01 | – | AB065426 | – | – | – |
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Bo-01 | – | AB065428 | – | – | – |
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Heteroepichloe sasae | E.sasae-H | – | AB065432 | – | – | – |
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E.sasae-N | – | AB065431 | – | – | – |
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Keithomyces carneus | CBS 239.32 | EF468988 | NR_131993 | NG_057769 | EF468938 | EF468789 |
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Keithomyces sp. | CBS 126563 | MT078871 | MT078883 | MT078856 | MT078921 | – |
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Marquandomyces marquandii | CBS 182.27 | EF468990 | MH854923 | MH866418 | EF468942 | EF468793 |
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Marquandomyces sp. | CBS 127132 | MT078872 | MT078882 | MT078857 | MT078922 | – |
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Metapochonia bulbillosa | JCM 18596 | AB758252 | AB709836 | AB709809 | AB758690 | AB758460 |
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CBS 145.70 | AF339591 | MH859529 | AF339542 | EF468943 | EF468796 |
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Metapochonia cordycipiticonsociata | CGMCC 3.17365 | KM263572 | KM263569 | KM263573 | KM263579 | KM263584 |
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CGMCC 3.17366 | KM263570 | KM263567 | KM263574 | KM263580 | KM263582 |
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Metapochonia goniodes | CBS 891.72 | AF339599 | AJ292409 | AF339550 | DQ522458 | DQ522354 |
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Metapochonia microbactrospora | CBS 101433 | – | AJ292408 | AF339538 | KJ398701 | KJ398794 |
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Metapochonia rubescens | CBS 464.88 | AF339615 | MH862138 | MH873830 | EF468944 | EF468797 |
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JCM 18620 | AB758247 | AB709859 | AB709832 | AB758685 | AB758455 |
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Metapochonia suchlasporia var. catenata | CBS 248.83 | – | MH861579 | MH873310 | KJ398696 | KJ398789 |
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CBS 251.83 | – | MH861580 | MH873311 | KJ398697 | KJ398790 |
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Metarhiziopsis microspora | CEHS133a | – | EF464589 | EF464571 | – | – |
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INEHS133a | – | EF464583 | EF464572 | – | – |
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Metarhizium anisopliae | ARSEF 7487 | – | HQ331446 | – | DQ468370 | DQ463996 |
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CBS 130.71 | MT078868 | MT078884 | MT078853 | MT078918 | MT078845 |
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Metarhizium flavoviride | CBS 125.65 | MT078869 | MT078885 | MT078854 | MT078919 | MT078846 |
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CBS 700.74 | MT078870 | – | MT078855 | MT078920 | MT078847 |
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CBS 218.56 | – | MH857590 | MH869139 | KJ398694 | KJ398787 |
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Moelleriella phyllogena | CUP 067785 | – | – | EU392610 | – | EU392674 |
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CUP 067793 | – | – | EU392608 | – | EU392672 |
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Moelleriella umbospora | CUP 067817 | – | – | EU392628 | – | EU392688 |
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Morakotia fusca | BCC 64125 | – | – | KY794862 | – | KY794857 |
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BCC 79272 | – | – | KY794861 | – | KY794856 |
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Mycophilomyces periconiae | CPC 27558 | – | KY173418 | KY173509 | – | – |
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Myriogenospora atramentosa | A.E.G 96-32 | AY489701 | – | AY489733 | DQ522455 | AY489628 |
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Neoaraneomyces araneicola | DY101711 | – | MW730520 | MW730609 | MW753026 | MW753033 |
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DY101712 | – | MW730522 | MW730610 | MW753027 | MW753034 |
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Neobarya parasitica | Marsons/n | – | KP899626 | KP899626 | – | – |
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Niesslia exilis | CBS 560.74 | AY489688 | MG827005 | AY489720 | – | AY489614 |
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Nigelia aurantiaca | BCC 13019 | GU979939 | – | GU979948 | GU979971 | GU979957 |
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Nigelia martialis | EFCC 6863 | – | – | JF415974 | – | JF416016 |
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Orbiocrella petchii | NHJ 6209 | EU369104 | JN049861 | EU369039 | EU369081 | EU369023 |
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NHJ 6240 | EU369103 | – | EU369038 | EU369082 | EU369022 |
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Papiliomyces liangshanensis | EFCC 1452 | EF468962 | – | EF468815 | – | EF468756 |
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EFCC 1523 | EF468961 | – | EF468814 | EF468918 | EF468755 |
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Papiliomyces shibinensis | GZUH SB13050311 | KR153588 | KR153585 | – | – | KR153589 |
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Parametarhizium changbaiense | CGMCC 19143 | MN590231 | MN589741 | MN589994 | MT921829 | MN908589 |
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Parametarhizium hingganense | CGMCC 19144 | MN055706 | MN055703 | MN061635 | MT939494 | MN065770 |
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Paraneoaraneomyces sinensis | ZY 22.006 | OQ709248 | OQ709254 | OQ709260 | OQ719621 | OQ719626 | This study |
ZY 22.007 | OQ709249 | OQ709255 | OQ709261 | OQ719622 | OQ719627 | This study | |
ZY 22.008 | OQ709250 | OQ709256 | OQ709262 | OQ719623 | OQ719628 | This study | |
Parepichloe cinerea | Ne-01 | – | AB065425 | – | – | – |
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Periglandula ipomoeae | IasaF13 | – | – | – | KP689517 | KP689568 |
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Pochonia boninensis | JCM 18597 | AB758255 | AB709858 | AB709831 | AB758693 | AB758463 |
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Pochonia chlamydosporia | CBS 101244 | DQ522544 | JN049821 | DQ518758 | DQ522424 | DQ522327 |
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Pochonia chlamydosporia var. catenulata | CBS 504.66 | AF339593 | AJ292398 | AF339544 | EF469120 | EF469069 |
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Pochonia chlamydosporia var. catenulata | JCM 18598 | AB758248 | AB709837 | AB709810 | AB758686 | AB758456 |
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JCM 18600 | AB758266 | AB709839 | AB709812 | AB758704 | AB758474 |
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Pochonia chlamydosporia var. chlamydosporia | JCM 18605 | AB758261 | AB709844 | AB709817 | AB758699 | AB758469 |
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JCM 18607 | AB758270 | AB709846 | AB709819 | AB758708 | AB758478 |
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Pochonia chlamydosporia var. ellipsospora | JCM 18609 | AB758257 | AB709848 | AB709821 | AB758695 | AB758465 |
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JCM 18611 | AB758265 | AB709850 | AB709823 | AB758703 | AB758473 |
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Pochonia chlamydosporia var. spinulospora | JCM 18613 | AB758258 | AB709854 | AB709827 | AB758696 | AB758466 |
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JCM 18619 | AB758272 | AB709857 | AB709830 | AB758710 | AB758480 |
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Pochonia globispora | CBS 203.86 | – | MH861942 | MH873631 | – | – |
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Pochonia sinensis | ZY 22.009 | OQ709251 | OQ709257 | OQ709263 | OQ719624 | OQ719629 | This study |
ZY 22.010 | OQ709252 | OQ709258 | OQ709264 | OQ719625 | OQ719630 | This study | |
Pseudometarhizium araneogenum | DY101741 | – | MW730532 | MW730618 | MW753030 | MW753037 |
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DY101801 | – | MW730536 | MW730623 | MW753032 | MW753039 |
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Pseudometarhizium lepidopterorum | SD05361 | – | MW730543 | MW730624 | – | MW753041 |
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SD05362 | – | MW730611 | MW730629 | – | MW753042 |
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Purpureomyces khaoyaiensis | BCC 1376 | KX983468 | – | KX983462 | KX983465 | KX983457 |
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Purpureomyces maesotensis | BCC 89300 | – | MN781917 | MN781876 | – | MN781733 |
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BCC 88441 | – | MN781916 | MN781877 | MN781824 | MN781734 |
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Purpureomyces pyriformis | BCC 85074 | – | MN781929 | MN781873 | MN781821 | MN781730 |
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Regiocrella camerunensis | ARSEF 7682 | – | – | DQ118735 | – | DQ118743 |
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Rotiferophthora angustispora | CBS 101437 | AF339584 | AJ292412 | AF339535 | DQ522460 | AF543776 |
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Samuelsia chalalensis | CUP 067856 | – | – | EU392637 | – | EU392691 |
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Samuelsia mundiveteris | BCC 40021 | – | – | GU552152 | – | GU552145 |
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Samuelsia rufobrunnea | CUP 067858 | – | – | AY986918 | – | AY986944 |
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Shimizuomyces paradoxus | EFCC 6279 | EF469131 | JN049847 | EF469084 | EF469117 | EF469071 |
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EFCC 6564 | EF469130 | EF469083 | EF469118 | EF469072 |
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Sungia yongmunensis | EFCC 2131 | EF468977 | JN049856 | EF468833 | – | EF468770 |
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EFCC 2135 | EF468979 | – | EF468834 | – | EF468769 |
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Tyrannicordyceps fratricida | TNS 19011 | JQ257022 | – | JQ257023 | JQ257021 | JQ257028 |
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Ustilaginoidea dichromenae | MRLIB 9228 | – | – | – | JQ257018 | JQ257025 |
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Ustilaginoidea virens | ATCC 16180 | – | – | – | JQ257019 | JQ257026 |
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MAFF 240421 | – | JQ349068 | JQ257011 | JQ257017 | JQ257024 |
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Yosiokobayasia kusanagiensis | TNS-F18494 | JF415954 | JN049873 | JF415972 | – | JF416014 |
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Pleurocordyceps aurantiaca | MFLUCC 17-2113 | MG136904 | MG136916 | MG136910 | MG136870 | MG136875 |
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Pleurocordyceps marginaliradians | MFLU 17-1582 | MG136908 | MG136920 | MG136914 | MG271931 | MG136878 |
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Lasergene software (version 6.0, DNASTAR) was used to analyse the ambiguous bases of the PCR amplicon sequences. The SSU, ITS, LSU, RPB2 and EF1A sequences were retrieved from the GenBank database, based on previous studies by
In the present study, the combined loci were analysed using the Bayesian Inference (BI) and the Maximum Likelihood (ML) methods. MrBayes version 3.2 (
Pleurocordyceps aurantiacus (MFLUCC 17-2113) and P. marginaliradians (MFLU 17-1582) were used as the outgroup for the phylogenetic analysis. The concatenated sequences (SSU, ITS, LSU, RPB2 and EF1A) included 113 taxa and consisted of 3,368 nucleotides (SSU, 905 bp; ITS, 448 bp; LSU, 453 bp; RPB2, 756 bp; and EF1A, 806 bp) with inserted gaps (Suppl. material
The phylogenetic trees (Fig.
Phylogram based on the Maximum Likelihood (ML) analysis using the SSU, ITS, LSU, RPB2 and EF1A sequences of Clavicipitaceae. The ML bootstrap values (≥ 70%) and the Bayesian posterior probability values (≥ 0.70) are indicated along the branches (BP/ML). The new taxa are highlighted in bold.
Based on its close phylogenetic relationship to Neoaraneomyces.
China.
Saprobic in soil. Sexual morph: not observed. Asexual morph: Hyphae hyaline, smooth, branched, septate. Phialides arising from aerial hyphae or hyphae regimental, solitary, straight to flexuous, tapering with enlarged base, smooth, hyaline. Conidia borne on the apex of the phialides or in small globose heads at the apices of the phialides. Conidia cymbiform to reniform, smooth-walled, one-celled, adhering in globose heads or the apex of phialides.
Paraneoaraneomyces sinensis Zhi. Y. Zhang & Y. F. Han.
Currently, the family Clavicipitaceae includes 52 genera and more than 500 species (
After the country of origin.
Kaili City, Guizhou Province, China; 27°17’56’’N, 108°82’68’’E; isolated from the green belt soil in July 2022; Zhi-Yuan Zhang (holotype ZY H-22.006, ex-holotype ZY 22.006, ibid., ZY 22.007).
Guizhou Province, China.
Culture characteristics (14 days at 25 °C): Colonies on PDA 35–37 mm in diameter, white, slightly raised at centre, fluffy, nearly round, margin regular; reverse: pale yellow. Colonies on MEA 35–37 mm in diameter, white, plicated, flocculent, nearly round, margin regular; reverse: pale yellow. Colonies on SNA 29–31 mm in diameter, white, flat, felty, nearly round, margin regular; reverse: white, compact at centre. Colonies on OA 36–38 mm in diameter, white, felty, early round, margin regular; reverse: white.
Hyphae hyaline, smooth, branched, septate, 1.0–3.0 μm in diameter. Phialides arising from aerial hyphae or hyphae regimental, solitary, straight to flexuous, tapering with enlarged base, smooth, hyaline, 19.0–34.0 × 0.5–1.5 µm (av. 27.0 × 1.1, n = 50). Conidia borne on the apices of the phialides or in small globose heads at the apex of the phialides. Conidia cymbiform to reniform, smooth-walled, one-celled, adhering in globose heads or the apex of phialides, 3.0–5.5 × 1.0–1.5 µm (av. 4.3 × 1.4, n = 50). Sexual morph undetermined.
Kaili City, Guizhou Province, China; 27°17’72’’N, 108°83’10’’E; isolated from the green belt soil in July 2022; Zhi-Yuan Zhang, ZY 22.008.
The multi-locus phylogenetic analyses showed that Paraneoaraneomyces sinensis is closely related to Neoaraneomyces araneicola (Fig.
After the country of origin.
Kaili City, Guizhou Province, China; 27°17’56’’N, 108°82’68’’E; isolated from the green belt soil in July 2022; Zhi-Yuan Zhang (holotype ZY H-22.009, ex-holotype ZY 22.009, ibid., ZY 22.010).
Guizhou Province, China.
Culture characteristics (14 days at 25 °C): Colonies on PDA fast-growing, reaching 74–77 mm in diameter, white, flat, fluffy to flocculent, margin identified; reverse: white. Colonies on MEA 67 mm in diameter, white, flat, compact, fluffy to flocculent, margin identified; reverse: white. Colonies on SNA 59–60 mm in diameter, white, aerial mycelia sparse, flat, flocculent, nearly round, margin regular; reverse: white. Colonies on OA 58 mm in diameter, white, aerial mycelia sparse, flat, felty, nearly round; reverse: white.
Hyphae hyaline, smooth, branched, septate, 0.5–1.5 μm in diameter. Phialides produced from prostrate aerial hyphae, solitary or rarely in whorls of 2–3, slender, tapering towards the tip, 5.5–51.0 × 0.5–1.5 µm (av. 22.0 × 1.0, n = 50). Conidia in small globose heads at the apex of the phialides. Conidia ovoid, sometimes subglobose or ellipsoidal, smooth-walled, one-celled, adhering in globose heads, 3.0–4.5 × 2.0–3.0 µm (av. 3.6 × 2.5, n = 50). Swollen hyphae not observed. Dictyochlamydospores not observed. Crystals absent. Sexual morph undetermined.
The multi-locus phylogenetic analyses (Fig.
In this study, we proposed a new Pochonia species and a new genus Paraneoaraneomyces within the family Clavicipitaceae. This study has important implications for the species diversity, taxonomy and geographic distribution of Clavicipitaceae (Hypocreales).
Fungi are highly abundant eukaryotes (
Soil is the largest natural reservoir of microorganisms and is inhabited by a large number of fungi. Taxonomy of soil fungi is an emerging area of research. Currently, only about 800,000 species of soil fungi have been identified worldwide (
1 | Host is a plant | 2 |
– | Host insects, nematodes, rotifers, protozoans or soil | 3 |
2 | Asexual morph produced | Metarhiziopsis |
– | Sexual morph produced | 4 |
3 | Conidia with adhesive hapteron | Pseudomeria |
– | Conidia without adhesive hapteron | 5 |
4 | Stromata stalked | Neocordyceps |
– | Stromata lacking stalks | 6 |
5 | Conidia cymbiform to reniform | Paraneoaraneomyces |
– | Conidia fusiform or ellipsoidal | 7 |
6 | Host bamboo | Loculistroma |
– | Host grasses | Nigrocornus |
7 | Conidiophores mononematous | Neoaraneomyces |
– | Conidiophores synnematous or mononematous | Pseudometarhizium |
We greatly appreciate Dr. Bensch for her advice on the new species names. We appreciate Charlesworth for the English language editing to the whole manuscript.
No conflict of interest was declared.
No ethical statement was reported.
This work was financially supported by grants from the Guizhou Provincial Science and Technology Projects (ZK[2023]155), the Science Research Youth Program in Colleges and Universities (Qiankeji[2022]153), and the National Natural Science Foundation of China (no. 32060011, 31860520).
The individual contributions are as follows: Zhi-Yuan Zhang, Yao Feng, Shuo-Qiu Tong, and Chen-Yu Ding. conceptualized the study, performed microscopical examinations of fungal specimens, wrote, edited, and reviewed the manuscript. Zhi-Yuan Zhang and Yao Feng conducted phylogenetic studies. Gang Tao and Yan-Feng Han wrote, reviewed, and edited the manuscript. Shuo-Qiu Tong and Chen-Yu Ding prepared figures. Zhi-Yuan Zhang reviewed the manuscript and provided funding. All authors have read and agreed to the published version of the manuscript.
Zhi-Yuan Zhang https://orcid.org/0000-0003-2031-7518
Yao Feng https://orcid.org/0000-0002-0888-8775
Shuo-Qiu Tong https://orcid.org/0009-0006-9422-5551
Chen-Yu Ding https://orcid.org/0000-0003-2639-3300
Gang Tao https://orcid.org/0000-0002-0882-3752
Yan-Feng Han https://orcid.org/0000-0002-8646-3975
All of the data that support the findings of this study are available in the main text or Supplementary Information.
Sequence dataset
Data type: sequence
The best-fit evolutionary model in the phylogenetic analyses
Data type: table